Maternal and paternal anxiety during pregnancy: Comparing the effects on behavioral problems in offspring

Prenatal maternal anxiety has been associated with both short and long-term mental health problems in the child. The current study aims to examine the association between maternal and paternal prenatal anxiety and behaviour problems in the child at 1.5 and 5 years, using three different approaches; (1) adjusting for covariates, (2) using fathers’ anxiety during pregnancy as a negative control, and (3) using a sibling-comparison design, controlling for unmeasured family factors. We used data from the Norwegian Mother, Father and Child Cohort Study (MoBa) is used. MoBa is a cohort consisting of about 114 000 pregnancies (about 34000 siblings) recruited from 1999 to 2008. Self-reported measures on maternal anxiety were obtained twice in pregnancy and 6 months after birth, while paternal anxiety was reported prenatally at 17th weeks of gestation. Maternal reports on child behaviour problems were obtained at 1.5 and 5 years of age. Results suggests that prenatal exposure to maternal anxiety was associated with behaviour problems at 1.5 years: adjusted beta (β) = 0.13 (CI = 0.12, 0.15), and at 5 years: β = 0.11 (CI = 0.09, 0.14). However, paternal anxiety was also associated with behaviour problems at 1.5 years: β = 0.03 (CI = 0.01–0.03) and at 5 years β = 0.03 (CI = 0.02, 0.03). These associations were attenuated in the sibling comparison analyses: β = -0.02 (CI = -0.02–0.05) at 1.5 years and β = -0.05 (CI = -0.10, 0.02) at 5 years. In conclusions, the sibling analyses are not consistent with a direct effect of prenatal maternal anxiety on child behaviour problems. It is more likely that genetic or shared family environment explain this association.


Introduction
Exposure to maternal stress during pregnancy, such as anxiety, is a known risk factor for a wide range of developmental outcomes, and is associated with shortened gestation, restricted fetal growth, and increased emotional and behavioral difficulties in offspring [1][2][3]. The mechanism linking maternal exposures during pregnancy and offspring outcomes has been there was a direct effect of prenatal maternal depression and anxiety, most of the observed effects could be explained by residual confounding (i.e., either paternal symptoms or postnatal effects of parental symptoms). However, they did not examine behavior problems. A third approach to account for confounding factors is to apply a sibling-comparison design. This approach may provide an effective control for familial factors that are constant (maternal genes and family context). Two large-scale studies that compared siblings differentially exposed prenatally to depression [19] or adverse life-events [20] did not find any association to offspring behavior. Similar findings emerged when the association between maternal prenatal anxiety and child emotional difficulties at 6 and 36 months was examined using a sibling design [21]. These studies found that the association was confounded by shared family effects. However, as maternal prenatal anxiety may differentially relate to child internalizing versus externalizing versus neurodevelopmental outcomes [18,22], the biological pathway from prenatal exposure to maternal anxiety on child behaviour may differ depending on the specific outcome. Based on these previous studies, maternal prenatal anxiety seems to have a stronger influence on the development of externalizing difficulties (i.e. behaviour problems) compared to internalizing ones (i.e. emotional difficulties) [18,22]. Studies also suggest that the effect of exposure to maternal prenatal anxiety may not occur in infancy but at a later age [23].
Building upon this earlier evidence, in the present study we examine whether the association between maternal prenatal anxiety and child behaviour problems in early childhood can be attributed to measured (known covariates, paternal prenatal anxiety) and unmeasured (shared genetic variance and environment) confounding. We aim to examine the association between maternal prenatal anxiety and behavior problems, using three different approaches: (i) adjusting for covariates and postnatal anxiety as a negative control, (ii) using fathers' anxiety during pregnancy as a negative control, and (iii) using a sibling-comparison design, controlling for unmeasured family factors. We aim to examine effects both in infancy and later in childhood by using behavior problems at both 1.5 and 5 years as the outcome.

Materials and methods
This study is a subproject of the Norwegian Mother, Father, and Child Cohort Study (MoBa) conducted by the Norwegian Institute of Public Health [24]. MoBa is a cohort comprising 114,247 pregnancies recruited from 1999 to 2008 with a participation rate of 40.6% [25]. All but two of the 52 hospitals across Norway agreed to participate in the recruitment for the study. Women were invited to participate when they attended the routine ultrasound examination that is offered to all pregnant women at 17-18 weeks of gestation (www.fhi.no/moba-en). The maternal-questionnaire response rates at the 17 th and 30 th weeks of gestation, and at 1.5 and 5 years after birth, were 95.1%, 91.4%, 87.0%, and 54.0%, respectively [25]. Written informed consent was obtained from all participating women. The Regional Committee for Medical and Health Research Ethics approved the study.
Within MoBa, 59,292 mothers of 68,602 children completed a series of questionnaires for each unique pregnancy, at the 17 th and 30 th weeks of gestation, and at 6 months and 1.5years after birth. The questionnaires included maternal anxiety level (17 th and 30 th weeks of gestation and 6 months after birth), paternal anxiety level (17 th week of gestation), and infantbehavior problems (1.5 and 5 years), along with other maternal characteristics such as age, education, marital status, smoking and drinking habits, and partner harmony. MoBa was also linked to the Medical Birth Registry of Norway (MBRN), which contains detailed medical information about newborn children (e.g., sex, birth weight, and gestational age) as well as their mothers (e.g., parity and birth complications).
In MoBa, there were 8,823 pairs of siblings at 1.5 years, and 3,428 of siblings at 5 years after birth with complete information. The mean age-difference between siblings was 2.7 years. The numbers dropped slightly after excluding half-siblings who had the same mother but different fathers (Fig 1). We used version 8 of the quality-assured data files; an overview of the questionnaires and items is available at https://www.fhi.no/en/studies/moba/for-forskere-artikler/ questionnaires-from-moba/.

Measures
Maternal and paternal anxiety. Mothers reported on symptoms of anxiety using six items from the validated short version of the Hopkins Symptom Checklist, two from SCL-5 and four from SCL-8 [26]. Items from the SCL are scored on a Likert scale ranging from 1 (not at all bothered) to 4 (very much bothered) and have been validated with a correlation of 0.92 with the SCL-25 [26]. Maternal anxiety was assessed twice during pregnancy (at the 17 th and 30 th gestational weeks) and once after birth (when the child was six months old). The mean scores for the 17 th and the 30 th gestational weeks were calculated separately using the items of constantly "frightened or anxious," "nervous, inner turmoil," "feel tense or stressed," and "sudden fear without reason." The maternal prenatal anxiety score was defined as the mean of the anxiety score at the 17 th and 30 th weeks. Paternal anxiety was also assessed once at the 17 th week of gestation using four items from the SCL-8.
Child-behavior problems. Child-behavior problems were measured by items from the Child Behavior Checklist (CBCL/TRF) [27]. A team of clinical and developmental psychologists selected the items from the CBCL used in MoBa. The selected items were based on theoretical and empirical representativeness and are representative with a correlation of 0.92 with the full scale [28]. Mothers responded to 8 items at 1.5 years and 10 items at 5 years on a threepoint Likert scale, from not true ("0") to sometimes true ("1") to often true ("2"), and mean values were computed for each time point. Cronbach's alpha was 0.57 and 0.74 for 1.5 years and 5 years, respectively. Potential confounders. The following variables were considered to be potential confounders: alcohol consumption during pregnancy (coded as never "0" and more than once a month "1"), smoking in pregnancy (coded as never "0," sometimes "1," and daily "2"), 10 items regarding partner harmony from the Marital Satisfaction Scale (MSS) with response options ranging from 1 to 6 (the higher, the worse), marital status (married/living together "0" and single "1"), and low maternal education (coded as higher university degree +4 years "0," college/university 3 years "1," college 1-2 years "2," and secondary school "3"). We also controlled for the following variables extracted from the MBRN: parity (coded as 0, 1, 2, 3, and 4 +), birth complications (coded as yes "1" or no "0"), and child sex (girl "0," boy "1"). The Pearson correlations between the variables can be found in Fig 2.

Statistical analyses
In the full cohort, the associations of maternal anxiety with child-behavior problems at 1.5 and 5 years were estimated using two different regressions: 1) ordinary linear regression of the child-behavior problem score on the maternal-anxiety score and 2) logistic regression of the dichotomized child-behavior problem score on the dichotomized maternal-anxiety score. The two regression models were adjusted for maternal smoking, alcohol consumption, parity, parental education, marital status, birth complication, and the interaction term between maternal prenatal and postnatal anxiety. When the outcome variable was the child-behavior problem measured at 5 years, the regressions were further adjusted for the child-behavior problem at 1.5 years.
Adjusted model: Additionally, we focused on the association between the maternal/paternal anxiety score at the 17 th week and the child-behavior problem. The child-behavior problem score was regressed on the maternal/paternal anxiety score at the 17 th week with adjustment for the paternal/maternal anxiety score at the 17 th week and the same set of adjusting variables used in the full cohort.
Mutually adjusted model: Fully adjusted model: In the sibling design, the association between maternal anxiety and the child-behavior problem was estimated with an adjustment for the family-shared effect. We used two types of regression: 1) regression of the inter-sibling difference in the child-behavior score on the inter-sibling difference in the maternal anxiety score and 2) conditional-logistic regression of the dichotomized child-behavior problem score on the dichotomized maternal-anxiety score (fixed effect) and family identification (random effect). The same set of adjusting variables mentioned above was used.

Missing data
Information about the proportion of missing data is given in Tables 1 and 2. Missing data in MoBa have previously been examined by Nilsen et al. [29]. Table 1 presents descriptive statistics for the full cohort. The mean score of child-behavior problems at 1.5 years increased with the severity of maternal prenatal and postnatal anxiety and paternal anxiety. The mean score of child-behavior problems at 1.5 years increased from 0.49 to 0.58 as maternal prenatal anxiety shifted from light to severe. Similarly, the mean score of child-behavior problems at 5 years increased from 0.35 to 0.51 with the severity of anxiety for the mothers measured postnatally (six months after birth). The mean score of child behavior at both 1.5 and 5 years also increased with the severity of paternal anxiety, but the inclination was not as large as that with maternal anxiety. Among the 18,767 siblings (Table 2) with complete data on child-behavior problems at 1.5 years, the mean score of child-behavior problems increased and decreased (0.47 ! 0.57 ! 0.48) as maternal prenatal anxiety escalated from light to moderate to severe. We observed a similar pattern in the mean score of child-behavior problems at 5 years. However, the mean score of child-behavior problems at both 1.5 and 5 years increased as maternal postnatal (or paternal) anxiety became severe.

Fathers as a negative control
We compared the effects of maternal anxiety and paternal anxiety at the 17 th week on childbehavior problems at 1.5 and 5 years (Table 4) because paternal anxiety was assessed at week 17 only. The effect size of paternal anxiety on child-behavior problems was smaller than that of maternal anxiety. The effect size of maternal anxiety on child-behavior problems at 1.5 years (0.06, 95% CI 0.06, 0.07) was twice the effect size of paternal anxiety (0.03, 95% CI: 0.02, 0.04). These maternal and paternal effects were reduced after adjustment for potential confounders, but remained significant.

Sibling comparisons
The associations between maternal anxiety and child-behavior problems were adjusted for family-shared effects in the sibling design ( Table 5). The moderate associations observed in the full cohort (Table 3) almost vanished in the sibling design. For example, the adjusted effect of prenatal anxiety on child behavior problems at 1.5 years was weak 0.02 (CI 95% -0.01, 0.05). The adjusted effect of prenatal anxiety on child-behavior problems at 5 years also appeared to be weak 0.02 (CI 95% -0.03, 0.05). Maternal prenatal and postnatal anxiety did not affect childbehavior problems after adjustment in this sibling design. We found consistent results from conditional logistic regressions.

Discussion
In this study, we applied three different approaches to control for unmeasured and measured confounding when examining the link between maternal anxiety during pregnancy and childbehavior problems. We found that behavior problems were higher when children had been exposed to maternal anxiety during pregnancy. Mothers' postnatal anxiety measured at six months was also associated with increased behavior problems at 1.5 and 5 years of age. Although the associations were somewhat weaker, they remained after adjusting for a number of potential confounders. The effect sizes are equivalent to those found in previous studies [9]. The second approach was to apply the father's anxiety measured during pregnancy as a negative control. Paternal anxiety operates as a negative control based on the assumption that Table 3. The effect of maternal anxiety on child behavior problems at 1.5 and 5 years (full cohort). paternal anxiety during pregnancy does not have any direct effect on the intrauterine environment coupled with the assumption that confounds are equally associated with both paternal and maternal anxiety [11]. We found that paternal prenatal anxiety was associated with child behavior. This suggests that the association between parental anxiety and child behavior could be confounded by shared environmental or genetic effects. However, this association was weaker than for maternal anxiety. This finding is in line with previous studies using paternal anxiety as a negative control [30] and points to the possibility of a direct biological effect of maternal anxiety through the intrauterine environment on child behavior [11].
To account for shared family effects, we applied a sibling-comparison design. In the sibling-comparison analyses, no associations were found for maternal anxiety measured either prenatally or at six months after birth. The implication is that maternal genetic and familyenvironmental factors that are shared by siblings may explain the consistent associations found in epidemiological studies examining the fetal-programming hypothesis. This finding is in line with previous studies examining relationships between depression during pregnancy and offspring mental health [16,19]. However, no previous studies have examined paternal anxiety as a negative control, the timing of exposure, and maternal anxiety and associations with short-and long-term child behavior.

Strengths and limitations
A major strength of this study was applying a sibling-comparison design. Sibling designs can reveal spurious effects of the explanatory variables, since many of these factors are similar through successive pregnancies and thus exposed to each sibling. While non-twin sibling designs cannot completely control for genetic effects, they do offer a relatively effective control for shared genetic influences as well as non-measured shared family confounds. However, an advantage of the non-twin sibling design over the MZ-twin design is that it provides an opportunity to examine different environmental exposures in utero.
Another example is extrapolating from animal models to complex human traits. Whether the stress response in rodents and humans shares similar mechanisms is subject to debate. This is particularly so in light of findings that imply the possibility that effects of stress during pregnancy in humans may be due to confounding factors not taken into consideration in animal research. Because we used data from a large population-based dataset, we were able to control for a number of covariates, such as birth complications, smoking and alcohol consumption during pregnancy, and maternal age and education. One of the limitations of our study is the potential lack of representation due to the selection bias of MoBa participants. However, a study examining the effect of recruitment bias using data from the MoBa study showed that even though prevalence estimates of exposures and outcomes were not always equal to that of the background population, estimates of associations were not biased [29]. The women participating with more than one pregnancy in the cohort might also represent an element of selection bias. As has been suggested by Sjölander et al., [30] there is a possibility that the first pregnancy influences the second. Additionally, since we used self-reports from the mothers, the associations may be biased by commonmethod variance. For instance, highly anxious mothers may be poor judges of their children's difficult behavior compared to mothers experiencing less anxiety. However, since we examine the outcomes of young children, mothers were deemed to be the individuals with the most knowledge about their child's behavior, outweighing the possibility of bias through halo effects. Using the same informant-the mother-is often unavoidable in large cohort studies. Furthermore, as the behaviors measured were mostly observable child behaviors, they seemed less liable to bias than other child traits might be.
A third limitation is that we used short scales with few items to measure anxiety when broader measures of anxiety and clinical diagnostic interviews would have been optimal. However, in large-scale studies with over 100,000 pregnancies, clinical interviews would be extremely time-consuming and costly. Furthermore, these items were found to correlate highly with the longer version of the shortened scale [28].
In sum, this study suggests that the well-established association found for prenatal maternal anxiety and child behavior difficulties was no longer present, once stable confounding factors was accounted for in a sibling comparison design. Our findings push forward our thinking about early experiences and precursors for child difficulties in the first five years of life. Pregnant women of today may feel more confident that fetal psychological experiences may be less risky than previously thought because we have improved the study design and methodology.

Conclusions
In this large prospective cohort, we found that both maternal and paternal anxiety were associated with behavior problems, after adjusting for multiple controls. However, this association was attenuated within full-sibling pairs. Our findings suggest that the association between prenatal maternal anxiety and behavioral difficulties is confounded by genetic and/or other family factors.